The field of the present invention is the magnification of images of objects, and more particularly microscopes and the field of microscopy.
Microscopy is used to produce magnified representations of both dynamic and stationary objects or samples. There are many different modes of microscopy such as brightfield microscopy, darkfield microscopy, phase contrast microscopy, fluorescence microscopy, reflectance or reflected light microscopy and confocal microscopy. All of these forms of microscopy deliver illumination light in a controlled fashion to the sample and collect as much of the light containing the desired information about the sample as possible. Typically, this is accomplished using Kohler illumination in any of reflectance microscopy, transmission microscopy or epifluorescence microscopy. Both of these methods use appropriately placed diaphragms and lenses to control both the size of the numerical aperture (illumination cone) and the size of the illuminated area of the sample. In Kohler illumination, diaphragms are placed in at least two locations. First, a diaphragm is placed in the conjugate image plane of the sample, a location which permits control of the size of the illuminated area of the sample. Second, a diaphragm is placed in the conjugate image plane of the aperture diaphragm of the objective lens(es) (this location is also a conjugate image plane of the aperture diaphragm of the condensor lens(es)), a location which permits control of the angle(s) of the light illuminating the sample. Typically, any of the diaphragms can be a simple iris (for example, for brightfield microscopy and epillumination fluorescence microscopy), but the diaphragms can also be more complex (for ex ample, in darkfield microscopy, where the diaphragms may comprise cutout rings of different diameters).
An example of a microscope using Kohler illumination is set forth in FIGS. 1A and 1B. In the figure, microscope 2 comprises a light source 4 that emits a plurality of light rays, which have been divided into first light rays 6, second light rays 8 and third light rays 10. The light rays are transmitted along an illumination light path 3 from light source 4 through light source lens 12, adjustable iris field diaphragm 14 and condenser lenses 16. An adjustable iris aperture diaphragm (condenser) 18 can be disposed between upstream and downstream condenser lenses 16. The light then contacts, or impinges upon, sample 20 and then proceeds to pass through objective lenses 22, which objective lenses can comprise an aperture diaphragm (objective) 24 spaced between the objective lenses 22, and then the light rays proceed to a light detector 26. As noted above, the angle of illumination of the sample can be controlled by modulating the light as it passes through conjugate image planes of the aperture diaphragm of the objective lens, which planes can be found, for example, at light source 4 and the upstream aperture diaphragm 18 in FIG. 1A, while the location and/or area of illumination of the sample can be controlled by modulating light as it passes through a conjugate image plane of the sample, which plane corresponds to the adjustable iris field diaphragm 14 in FIG. 1A.
One preferred form of microscopy is confocal microscopy, in which discrete aperture spots are illuminated in the object plane of the microscope from which transmission, reflected or fluorescent light is then relayed for observation through conjugate apertures in the image plane. In some embodiments, confocal microscopy can result in spatial resolution about 1.3 times better than the optimum resolution obtainable by conventional light microscopy. See, e.g., U.S. Pat. No. 5,587,832. Additionally, confocal microscopy can reduce the interference of stray, out-of-focus light from an observed specimen above or below the focal plane, and can permit optical sectioning of tissue as well as high-resolution 3-D reconstruction of the tissue. The technique can effectively resolve individual cells and living tissue without staining. Confocal microscopy can be performed using mechanical translation of the specimen with fixed optics, or using a fixed specimen and scanning beams manipulated by special rotating aperture disks. See, U.S. Pat. Nos. 4,802,748, 5,067,805, 5,099,363, 5,162,941. Such disks typically comprise a plurality of apertures, but only one aperture at a time is used for confocal scanning. Still other known confocal scanning systems have used a laser beam rastered with rotating mirrors to scan a specimen or a laser beam that scans a slit rather than a spot; such slit scanning increases imaging speed but slightly degrades resolution. See, U.S. Pat. No. 5,587,832.
Conventional confocal microscopes can be slow to acquire images for certain applications and become even slower as the scan line density increases and the aperture separation decreases. In addition, it is difficult to practically adjust the perimeters of the confocal microscope in commercial systems, and the signal to noise ratio (SNR) is sacrificed to increase the imaging rate. In addition, proper alignment of conventional confocal systems can be critical and difficult to maintain. In addition, laser-based systems are more expensive than white light systems, but such laser systems do not offer a selection of illumination wavelengths and can also lead to photo toxicity and rapid photo bleaching.
Thus, there has gone unmet a need for improved microscopy systems, including confocal microscopy systems, wherein the angle of illumination of a sample can be easily and rapidly controlled. There has also gone unmet a need for a microscope that can easily and rapidly control the quantity of light reaching the sample, including both varying the absolute quantity of light as well as the location on the sample upon which the quantity of light impinges. The present invention provides these and other advantages.
The present invention provides microscopes that have significant advantages in controlling the light that contacts a sample and/or that is detected emanating from a sample. The improved control includes enhanced, selective control of the angle of illumination, the quantity of light and the location of light reaching the sample and/or detector. The present invention provides these advantages by placing one or more spatial light modulators in the illumination and/or detection light path of the microscope at one or both of the conjugate image plane of the aperture diaphragm of the objective lens and the conjugate image plane of the sample.
Thus, in one aspect the present invention provides microscopes comprising a spatial light modulator comprising an illumination array of individual light transmission pixels, the spatial light modulator disposed in an illumination light path of the microscope at a conjugate image plane of an aperture diaphragm of an objective lens to provide an upstream spatial light modulator.
In some embodiments, the microscopes selectively control the angle of illumination of a sample and the angle of detection of light emanating from the sample, wherein the upstream spatial light modulator is operably connected to a modulator controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator to select a desired angle of illumination and detection of the sample and wherein a selected portion of the individual light transmission pixels corresponding to the desired angle of illumination and detection is on. In other embodiments, the microscopes selectively control a quantity of light reaching a sample, the quantity being less than all the light emitted by a light source located at a beginning of the illumination light path, wherein the upstream spatial light modulator is operably connected to a modulator controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator to select a desired quantity of illumination and a selected portion of the individual light transmission pixels corresponding to the desired quantity of illumination is on.
In further embodiments, the microscopes are transmission or reflectance microscopes. The microscopes can comprise a lens disposed in the illumination light path between a light source and the spatial light modulator, the light source disposed at a conjugate image plane of an aperture diaphragm of the objective lens that is located upstream from the upstream spatial light modulator. The microscopes can further comprise a second spatial light modulator that is disposed in a detection light path, located at a downstream conjugate image plane of an aperture diaphragm of the objective lens and operably connected to a second modulator controller containing computer-implemented programming that controls transmissive characteristics of the second spatial light modulator. Preferably, the first modulator controller is operably connected to the second modulator controller such that the second light modulator selectively controls the light in the detection light path to correspond to the desired angle selected by the first modulator controller; the various controllers discussed herein can be separate controllers or can be a single controller.
In still other embodiments, the microscopes provide darkfield microscopy, wherein the upstream spatial light modulator is operably connected to a first modulator controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator to select a desired pattern for darkfield microscopy and a selected portion of the individual light transmission pixels corresponding to the desired pattern for darkfield microscopy is on, and a second spatial light modulator is disposed in a detection light path, located at a downstream conjugate image plane of an aperture diaphragm of the objective lens and operably connected to a second modulator controller containing computer-implemented programming that controls transmissive characteristics of the second spatial light modulator to select a complementary pattern of the individual light transmission pixels of the second spatial light modulator to complement the pattern of individual light transmission pixels of the first spatial light modulator that are on, thereby providing a complementary pattern of the individual light transmission pixels of the second spatial light modulator that are on.
In more embodiments, the microscopes alternate between darkfield microscopy and brightfield microscopy. This can be achieved, for example, wherein the first modulator controller and the second modulator controller control the transmissive characteristics of the first and second spatial light modulators to switch between brightfield microscopy and at least one desired pattern for darkfield microscopy. For this and other embodiments that comprise cycling or a method, the microscopes preferably perform the method or cycle , for example, to alternate back and forth between darkfield microscopy and brightfield microscopy, with a cycle or method time of less than the time that is needed for the detector, such as a human eye or video camera, to acquire an image, for example less than about 0.03 seconds, thereby providing real-time viewing if desired. In other preferred embodiments, the microscopes alternate back and forth between darkfield microscopy and brightfield microscopy without refocusing.
In still more embodiments for this other aspects of the present invention (unless expressly stated otherwise or clear from the context, all embodiments of the present invention can be mixed and matched), the microscopes comprise a light detector located at a downstream end of a detection light path, the light detector comprising a detection array of individual detection pixels. In come embodiments, the detection array of individual detection pixels corresponds to and is aligned with the illumination array of individual light transmission pixels in the upstream spatial light modulator and the detection array of individual detection pixels is operably connected to a detector controller and the illumination array of individual light transmission pixels in the spatial light modulator is connected to the modulator controller, such that the modulator controller contains computer-implemented programming that selects a plurality of desired angles of illumination of the sample and the detector controller contains computer-implemented programming that detects the changes in intensity in the detection array of individual detection pixels corresponding to the plurality of desired angles of illumination and detection and therefrom determines a three-dimensional image of the sample. In other embodiments, which can be combined with the previous embodiments, the modulator controller selects a plurality of desired angles of illumination of the sample to provide a plurality of images of the sample at a corresponding plurality of different depths within the sample and a reconstruction controller comprises computer-implemented programming that tomographically reconstructs the different images to provide a three dimensional image of the sample.
In more embodiments, the modulator controller selects the plurality of desired angles of illumination of the sample such that the plurality of images of the sample at a corresponding plurality of different depths are obtained without moving the sample, a condenser lens or an objective lens.
In other embodiments, the light detector is a charge-coupled device, charge-injection device, video camera and the microscopes can comprise one or more photomultiplier tubes and or ocular eyepieces located at a downstream end of the detection light path. The spatial light modulator can be, for example, a digital micromirror device, microshutter or a liquid crystal device.
In another aspect, the present invention provides microscopes comprising a variable field iris, the microscopes comprising a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in an illumination light path at a conjugate image plane of a sample, to provide an upstream spatial light modulator, and that is operably connected to a modulator controller containing computer-implemented programming that controls transmissive characteristics of the upstream spatial light modulator to select a plurality of desired field iris settings. In some embodiments for this and other aspects of the invention, the individual light transmission pixels of the illumination array are in the on/off status corresponding to the desired field iris setting.
In a further aspect, the present invention provides microscopes that project a selected image into an image plane of a sample of the microscope, the microscope comprising a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in a first illumination light path at a conjugate image plane of the sample, to provide a first upstream spatial light modulator, wherein the upstream spatial light modulator is operably connected to at least one controller containing computer-implemented programming that varies an on/off status of the individual light transmission pixels of the illumination array to correspond to the selected image, the microscope further comprising a second illumination light path that provides illumination light to the sample. The selected image can be projected onto the sample or adjacent to the sample or elsewhere if desired.
In certain embodiments, the upstream spatial light modulator is disposed in both the first illumination light path and the second illumination light path and a first portion of the individual light transmission pixels of the illumination array provides illumination light to the sample and second portion of the individual light transmission pixels of the illumination array are in an on/off status corresponding to the selected image.
In further embodiments, wherein the microscopes firther comprise a light detector disposed downstream from the sample in a detection light path at a conjugate image plane of the sample, wherein the spatial light modulator and the light detector are operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the upstream spatial light modulator and that compiles the light intensity detection data provided by the light detector, the controller selectively varies the on/off status of individual light transmission pixels of the spatial light modulator to vary the light intensity impinging on selected spots of the sample and thereby vary the intensity of light emanating from the selected spots of the sample and impinging on at least one pixel of the light detector.
In a further aspect, the present invention provides microscopes that provide a uniform intensity of illumination light to a sample, the microscopes comprising a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in an illumination light path at a conjugate image plane of the sample, to provide an upstream spatial light modulator, and a light detector comprising a detection array of individual detection pixels and disposed downstream from the upstream spatial light modulator at a conjugate image plane of the sample and that receives light directly from the upstream spatial light modulator, wherein the spatial light modulator and the light detector are operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the upstream spatial light modulator and that compiles the light intensity detection data provided by the light detector, wherein the at least one controller selectively varies the on/off status of individual light transmission pixels of the spatial light modulator to compensate for non-uniform light intensities detected by the light detector, thereby providing a uniform intensity of illumination light that is transmitted to the sample.
In still another aspect, the present invention provides microscopes that vary the intensity of light emanating from a sample to a light detector, the microscopes comprising a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in an illumination light path at a conjugate image plane of the sample, to provide an upstream spatial light modulator, and the light detector which comprises a detection array of individual detection pixels and is disposed downstream from the sample in a detection light path at a conjugate image plane of the sample, wherein the spatial light modulator and the light detector are operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the upstream spatial light modulator and that compiles the light intensity detection data provided by the light detector, wherein the controller selectively varies the on/off status of individual light transmission pixels of the spatial light modulator to vary the light intensity impinging on selected spots of the sample and thereby vary the intensity of light emanating from the selected spots of the sample and impinging on at least one pixel of the light detector.
In various embodiments, the controller selectively varies the on/off status of individual light transmission pixels of the spatial light modulator such that when the light intensity impinging on a pixel of the light detector is greater than or equal to a threshold level that indicates that the light intensity significantly adversely affects adjacent pixels then at least one corresponding pixel in the upstream spatial light modulator is turned off for a time sufficient to reduce the light intensity impinging on the pixel of the light detector below the threshold level. In further embodiments, the controller selectively varies the on/off status of individual light transmission pixels of the spatial light modulator such the light intensity impinging on the detection array of pixels of the light detector is uniform across the detection array, and the controller determines the light intensity characteristics of the sample by determining an amount of time individual pixels in the illumination array of the upstream spatial light modulator are on or off.
In still further embodiments, the microscopes further comprise a second spatial light modulator that is disposed in the detection light path, located at a downstream conjugate image plane of a sample and operably connected to a second modulator controller containing computer-implemented programming that controls transmissive characteristics of the second spatial light modulator. In other embodiments, the microscopes can be a confocal microscope. For some embodiments, the detection array of individual detection pixels corresponds to and is aligned with the illumination array of individual light transmission pixels in the upstream spatial light modulator. The at least one controller contains computer-implemented programming that controls transmissive characteristics of the upstream spatial light modulator to provide at least one illumination spot to the sample by causing at least one individual light transmission pixel of the upstream spatial light modulator to be on while adjacent pixels are off and the detection array selectively detects the illumination spot. In preferred embodiments, the at least one controller causes the upstream spatial light modulator to simultaneously form a plurality of the illumination spots and to provide sequential complementary patterns of the spots.
In still other embodiments, the at least one controller causes the light detector to detect only light impinging on a central detection pixel aligned with and corresponding to an individual light transmission pixel of the upstream spatial light modulator that is on. The at least one controller can also cause the light detector to detect light impinging on a central detection pixel aligned with an individual light transmission pixel of the upstream spatial light modulator that is on and to detect light impinging on at least one detection pixel adjacent to the central detection pixel, and the controller contains computer-implemented programming that compiles the data provided by the at least one adjacent detection pixel and combines it with the data provided by the central detection pixel. In certain preferred embodiments the computer-implemented programming compiles the data provided by the adjacent detection pixels and combines it with the data provided by the central detection pixel such that the rejection of the out of focus information of the microscope is enhanced compared to the focus without the data from the adjacent detection pixels and/or compiles the data provided by the adjacent detection pixels and combines it with the data provided by the central detection pixel to determine the light absorption characteristics of the sample.
In still a further aspect, the present invention provides microscopes comprising a first spatial light modulator comprising a first illumination array of individual light transmission pixels, the first spatial light modulator disposed in an illumination light path at a conjugate image plane of an aperture diaphragm of an objective lens to provide a first upstream spatial light modulator and a second spatial light modulator comprising a second illumination array of individual light transmission pixels and disposed in the illumination light path at a conjugate image plane of a sample to provide a second upstream spatial light modulator. In further embodiments, the microscopes further comprise a third spatial light modulator disposed at the downstream conjugate image plane of an aperture diaphragm of the objective lens and/or a fourth spatial light modulator is disposed in the detection light path and located at the downstream conjugate image plane of a sample, as well as desired detectors and/or controllers.
In some embodiments, the microscopes provide confocal properties and selectively control the angle of illumination of a sample and the angle of detection of light emanating from the sample, wherein the computer-implemented programming that controls transmissive characteristics of the first spatial light modulator selects a desired angle of illumination and detection of the sample and wherein a selected portion of the individual light transmission pixels of the first spatial light modulator corresponding to the desired angle of illumination and detection is on and wherein the computer-implemented programming that controls transmissive characteristics of the second spatial light modulator provides at least one illumination spot to the sample by causing at least one individual light transmission pixel of the second spatial light modulator to be on while adjacent pixels are off and the detection pixels of the detection array are positioned to selectively detect the illumination spot.
In further embodiments, the computer-implemented programming causes the illumination array of individual light transmission pixels in the first spatial light modulator to provide a plurality of desired angles of illumination of the sample and the detection array of individual detection pixels to detect changes in intensity in the detection array of individual detection pixels corresponding to the plurality of desired angles of illumination and therefrom determining a three-dimensional image of the sample. The computer-implemented programming can also cause the illumination array of individual light transmission pixels in the first spatial light modulator to provide a plurality of desired angles of illumination of the sample to provide a plurality of images of the sample at a corresponding plurality of different depths within the sample and then tomographically reconstructs the different images to provide a three dimensional image of the sample. These embodiments are preferably effected without moving the sample, a condenser lens or an objective lens and can also selectively provide darkfield microscopy by causing the illumination array of individual light transmission pixels in the first spatial light modulator to provide a desired pattern for darkfield microscopy and a selected portion of the individual light transmission pixels corresponding to the desired pattern for darkfield microscopy is on and a complementary pattern of the individual light transmission pixels of the third spatial light modulator is on. The microscopes can alternate between darkfield microscopy and brightfield microscopy without refocusing.
In still further embodiments, the computer-implemented programming causes the light detector to detect only light impinging on a central detection pixel aligned with and corresponding to an individual light transmission pixel of the second spatial light modulator that is on, and can compile the data provided by the at least one adjacent detection pixel and combine it with the data provided by the central detection pixel. Thus the computer-implemented programming can compile the data provided by the adjacent detection pixels and combine it with the data provided by the central detection pixel such that the focus of the microscope is enhanced compared to the focus without the data from the adjacent detection pixels.
In yet further embodiments, the microscopes provide time-delayed fluorescence detection, wherein the computer-implemented programming causes at least one of the spatial light modulators to illuminate the sample for an amount of time suitable to induce fluorescence in the sample and then ceasing illuminating the sample, and then causing the detector to begin detecting fluorescence emanating from the sample.
In yet other aspects, the present invention provides microscopes comprising means for spatial light modulation disposed in an illumination light path at a conjugate image plane of an aperture diaphragm of an objective lens, which means for spatial light modulation can comprise means for selectively controlling a desired angle of illumination of a sample, wherein the means for spatial light modulation is operably connected to a computer means for controlling transmissive characteristics of the spatial light modulator for selecting the desired angle of illumination of the sample. The microscopes can comprise a variable field iris, the microscopes comprising means for spatial light modulation that is disposed in an illumination light path at a conjugate image plane of a sample, wherein the comprising means for spatial light modulation is operably connected to computer means for controlling transmissive characteristics of the spatial light modulator and for selecting a plurality of desired field iris settings.
In still yet other aspects, the present invention provides microscopes comprising that project a selected image into the image plane of a sample, the microscope comprising means for spatial light modulation that is disposed in an illumination light path at a conjugate image plane of the sample, wherein the means for spatial light modulation is operably connected to at least one computer means for controlling transmissive characteristics of the means for spatial light modulation and for projecting the selected image into the image plane of the sample, the microscope further comprising a second illumination light path that provides illumination light to the sample.
In other aspects, the present invention provides microscopes that provide a uniform intensity of illumination light to a sample, the microscope comprising means for spatial light modulation that is disposed in an illumination light path at a conjugate image plane of the sample, and means for detecting light that is disposed downstream from the means for spatial light modulation at a conjugate image plane of the sample and that receives light directly from the means for spatial light modulation, wherein the means for spatial light modulation and the means for detecting light are operably connected to at least one computer means for controlling transmissive characteristics of the means for spatial light modulation and that compiles the light intensity detection data provided by the means for detecting light, wherein the computer means selectively controls the transmissive characteristics of the means for spatial light modulation to compensate for non-uniform light intensities detected by the light detector, thereby providing a uniform intensity of illumination light that is transmitted to the sample.
In yet other aspects, the present invention provides microscopes that vary the intensity of light emanating from a sample to a means for detecting light, the microscope comprising means for spatial light modulation that is disposed in an illumination light path at a conjugate image plane of the sample and means for detecting light that is disposed downstream from the sample in a detection light path at a conjugate image plane of the sample, wherein the means for spatial light modulation and the means for detecting light are operably connected to at least one computer means for controlling transmissive characteristics of the means for spatial light modulation and that compiles the light intensity detection data provided by the means for detecting light, wherein the computer means selectively controls the transmissive characteristics of the means for spatial light modulation to vary the light intensity impinging on selected spots of the sample and thereby vary the intensity of light emanating from the selected spots of the sample and impinging on at least one pixel of the light detector.
In some embodiments, the computer means selectively varies the transmissive characteristics of the means for spatial light modulation such that when the light intensity impinging on a pixel of the means for detecting light is greater than or equal to a threshold level that indicates that the light intensity significantly adversely affects adjacent pixels then the transmissive characteristics of the means for spatial light modulation are varied for a time sufficient to reduce the light intensity impinging on the pixel of the means for detecting light below the threshold level. In other embodiments, the computer means selectively varies the transmissive characteristics of the means for spatial light modulation such that the light intensity impinging on the means for detecting light is uniform across the means for detecting light, and the computer means determines the light intensity characteristics of the sample by determining variation of the transmissive characteristics of the means for spatial light modulation. The microscopes can further comprise a second means for spatial light modulation that is disposed in the detection light path at a downstream conjugate image plane of a sample and is operably connected to a second computer means for controlling transmissive characteristics of the second means for spatial light modulation.
In still yet further aspects, the present invention provides microscopes comprising a first means for spatial light modulation that is disposed in an illumination light path at a conjugate image plane of an aperture diaphragm of an objective lens and a second means for spatial light modulation that is disposed in the illumination light path at a conjugate image plane of a sample. The microscopes can firther comprise a third means for spatial light modulation is disposed at the downstream conjugate image plane of the aperture diaphragm of the objective lens, a fourth means for spatial light modulation is disposed in the detection light path and located at the downstream conjugate image plane of the sample, or means for detecting light located at a downstream end of the detection light path. All the means for spatial light modulation and the means for detecting light can be operably connected to at least one computer means for controlling the light transmissive or detection characteristics of the at least one means for spatial light modulation and the means for detecting light.
In still yet more aspects, the present invention provides methods of modulating an illumination light path within a microscope wherein the illumination light path comprises a spatial light modulator comprising an illumination array of individual light transmission pixels at a conjugate image plane of an aperture diaphragm of an objective lens, the method comprising modulating the illumination light path by selecting light transmissive characteristics of the spatial light modulator. The methods can further comprise transmitting light along the illumination light path such that the light passes the spatial light modulator and thereby modulating the light by the spatial light modulator.
In some embodiments, the methods further comprise selectively controlling an angle of illumination of a sample and an angle of detection of light emanating from the sample by turning on a selected portion of the individual light transmission pixels corresponding to the desired angle of illumination and detection. In other embodiments, the methods further comprise selectively controlling the quantity of light reaching a sample, the quantity being less than all the light emitted by a light source located at a beginning of the illumination light path, the methods comprise selecting a desired quantity of illumination by turning on a selected portion of the individual light transmission pixels corresponding to the desired quantity of illumination. In further embodiments, the methods further comprise darkfield microscopy and the methods further comprise: setting on a selected portion of the individual light transmission pixels of the illumination array corresponding to a desired pattern for darkfield microscopy, and setting on a selected portion of the second array corresponding to a complementary pattern that complement the desired pattern of the illumination array. The methods can further comprise alternating between darkfield microscopy and brightfield microscopy with or without refocusing.
In still some other embodiments, wherein the spatial light modulator is connected to a modulator controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator and the microscope further comprises a light detector comprising a detection array of individual detection pixels and located at a downstream end of a detection light path, wherein the light detector is operably connected to a detector controller containing computer-implemented programming that controls detection characteristics of the detection array, and the methods further comprise: selecting a plurality of desired angles of illumination of the sample, and detecting the changes in intensity in the detection array corresponding to the plurality of desired angles of illumination, and therefrom determining a three-dimensional image of the sample. Alternatively, the methods can further comprise: selecting a plurality of desired angles of illumination of the sample to provide a plurality of images of the sample at a corresponding plurality of different depths within the sample, and tomographically reconstructing the different images to provide a three dimensional image of the sample. Preferably, the selecting and the reconstructing are performed without moving the sample, a condensor lens or an objective lens.
In still yet further aspects, the present invention provides methods for varying a field iris in a microscope wherein the microscope comprises a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in an illumination light path at a conjugate image plane of a sample, wherein the spatial light modulator is operably connected to a modulator controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator, the methods comprising: selecting a desired field iris setting by setting the individual light transmission pixels of the illumination array to an on/off status corresponding to the desired field iris setting, and then changing the individual light transmission pixels of the illumination array to a different on/off status corresponding to a different desired field iris setting, thereby varying the field iris.
In still more aspects, the present invention provides methods of projecting a selected image into an image plane of a sample of a microscope, the microscope comprising a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in a first illumination light path at a conjugate image plane of the sample, to provide a first spatial light modulator, wherein the first spatial light modulator is operably connected to at least one controller containing computer-implemented programming that varies an on/off status of the individual light transmission pixels of the illumination array to correspond to the selected image, the microscope further comprising a second illumination light path that provides illumination light to the sample, the method comprising: setting the individual light transmission pixels to an on/off status corresponding to the selected image, and projecting light onto the spatial light modulator such that the light passed by the spatial light modulator along the illumination light path to the image plane of the sample conveys the selected image. The methods can further comprise projecting the selected image onto the sample or adjacent to the sample.
In other embodiments, wherein the microscope further comprises a light detector comprising a detection array, the methods further comprise: selectively varying the on/off status of individual light transmission pixels of the spatial light modulator to vary the light intensity impinging on selected spots of the sample and thereby vary the intensity of light emanating from the selected spots of the sample and impinging on at least one pixel of the light detector.
In still other aspects, the present invention provides methods for providing uniform intensity of illumination light to a sample in a microscope, wherein the microscope comprises a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in an illumination light path at a conjugate image plane of a sample and a light detector comprising a detection array of individual detection pixels and disposed downstream from the upstream spatial light modulator at a conjugate image plane of the sample and that receives light directly from the upstream spatial light modulator, wherein the spatial light modulator and the light detector are operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the upstream spatial light modulator and that compiles the light intensity detection data provided by the light detector, the methods comprising: varying the on/off status of individual light transmission pixels of the spatial light modulator to compensate for non-uniform light intensities detected by the light detector, thereby providing a uniform intensity of illumination light that is transmitted to the sample.
In still more other aspects, the present invention provides methods for varying the intensity of light emanating from a sample to a light detector in a microscope, wherein the microscope comprises a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in an illumination light path at a conjugate image plane of the sample and a light detector that comprises a detection array of individual detection pixels and is disposed downstream from the sample in a detection light path at a conjugate image plane of the sample, wherein the spatial light modulator and the light detector are operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator and that compiles the light intensity detection data provided by the light detector, the methods comprising: varying an on/off status of individual light transmission pixels of the spatial light modulator to vary the light intensity impinging on selected spots of the sample and thereby varying the intensity of light emanating from the selected spots of the sample and impinging on at least one pixel of the light detector. In some embodiments, the methods can further comprise selectively varying the on/off status of individual light transmission pixels of the spatial light modulator such that when the light intensity impinging on a pixel of the light detector is greater than or equal to a threshold level that indicates that the light intensity significantly adversely affects adjacent pixels then turning off at least one corresponding pixel in the spatial light modulator for a time sufficient to reduce the light intensity impinging on the pixel of the light detector below the threshold level. The methods can also comprise selectively varying the on/off status of individual light transmission pixels of the spatial light modulator such the light intensity impinging on the detection array of pixels of the light detector is uniform across the detection array, and determining the light intensity characteristics of the sample by determining an amount of time individual pixels in the illumination array of the upstream spatial light modulator are on or off.
In still some other embodiments, wherein the microscope is a confocal microscope, the methods further comprise providing at least one illumination spot to the sample by causing at least one individual light transmission pixel of the spatial light modulator to be on while adjacent pixels are off and then the detection array selectively detects the illumination spot. Preferably, the spatial light modulator simultaneously forms a plurality of the illumination spots and provides sequential complementary patterns of the spots. In other embodiments, the methods further comprise detecting light impinging on a central detection pixel aligned with an individual light transmission pixel of the spatial light modulator that is on and detecting light impinging on at least one detection pixel adjacent to the central detection pixel, and compiling the data provided by the at least one adjacent detection pixel and combining it with the data provided by the central detection pixel. The methods can further comprise compiling the data provided by the adjacent detection pixels and combining it with the data provided by the central detection pixel such that the rejection of the out of focus information of the microscope is enhanced compared to the focus without the data from the adjacent detection pixels, or the methods can comprise compiling the data provided by the adjacent detection pixels and combining it with the data provided by the central detection pixel and therefrom determining the light absorption characteristics of the sample.
In some embodiments, the methods provide real time directly viewable confocal microscopy.
In other aspects, the present invention provides methods of confocal microscopy comprising use of a microscope comprising a first spatial light modulator comprising a first illumination array of individual light transmission pixels, the first spatial light modulator disposed in an illumination light path at a conjugate image plane of an aperture diaphragm of an objective lens to provide a first upstream spatial light modulator, a second spatial light modulator comprising a second illumination array of individual light transmission pixels and disposed in the illumination light path at a conjugate image plane of a sample to provide a second upstream spatial light modulator, and a light detector located at a downstream end of the detection light path, the light detector comprising a detection array of individual detection pixels, wherein the spatial light modulators and the light detector are operably connected to at least one controller containing computer-implemented programming that controls the light transmissive or detection characteristics of the spatial light modulators and the light detector, wherein the methods comprise: selecting a desired angle of illumination and detection by turning on a selected portion of the individual light transmission pixels of the first illumination array corresponding to the desired angle of illumination and detection, and selecting a desired illumination spot by turning on a selected portion of the individual light transmission pixels of the second illumination array corresponding to the desired illumination spot such at least one individual light transmission pixel of the second illumination array is on while adjacent pixels are off and the detection pixels of the detection array are positioned to selectively detect the illumination spot. Such methods can further provide 3-D imaging, with or without moving the sample, a condensor lens or an objective lens.
In more aspects, the present invention the methods comprise time-delayed fluorescence detection, the methods comprising: illuminating the sample for an amount of time suitable to induce fluorescence in the sample, ceasing illuminating the sample, and then detecting fluorescence emanating from the sample.
These and other aspects of the present invention will become evident upon reference to the following Detailed Description and attached drawings. In addition, various references are set forth herein that describe in more detail certain apparatus and methods (e.g., spatial light modulators, etc.); all such references are incorporated herein by reference in their entirety.